This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. We have recently discovered a novel regulator of glucose metabolism, the N-terminal dipeptidase, DPP2, a serine protease that had been identified and cloned by the Huber lab. We have generated a conditional DPP2 knock down (kd) mouse and crossed it with a neurogenin-3 (NGN3)-Cre transgenic (tg) mouse that led to specific kd of DPP2 in the hypothalamus of the brain and the gastrointestinal (GI) tract (NGN3-DPP2 kd). These mice spontaneously develop hyperinsulinemia, glucose intolerance and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in NGN3-DPP2 kd mice, which was associated with a significant increase in adipose tissue mass. This phenotype was exacerbated with age and when challenged with a high fat diet. We conclude, therefore, that DPP2 enzyme activity is essential for maintaining glucose homeostasis. This work constitutes an ongoing collaboration between two scientists on the Boston campus of Tufts: Brigitte T. Huber, PI, Department of Pathology, an immunologist who is an expert on DPP2 and has produced the conditional DPP2 kd mouse, and Ronald M. Lechan, co-PI, Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, a neuroendocrinologist and neuroanatomist who has characterized the DPP2 expression pattern in the brain. A joint publication has already resulted from the characterization of this new animal model for type 2 diabetes. In order to define whether the DPP2 enzyme activity controls glucose metabolism over a central (brain) or peripheral (GI tract) mechanism, we have recently crossed the conditional DPP2-loxP mice with Sf1-Cre tg mice, which leads to DPP2 kd selectively in the ventromedial nucleus (VMN) of the hypothalamus, but not the GI tract (Sf1-DPP2 kd). These mice develop similar glucose intolerance as the NGN3-DPP2 kd mice. Thus, we are now in an excellent position to identify the specific substrate(s) of DPP2 in the VMN that prevents hyperinsulinemia and obesity. We are applying for a Russo grant to lay the groundwork for this project, providing a solid basis for a dual investigator RO1 application to NIDDK that is planned for fall of this year (the specific aims are outlined in the summary section). Our main goal is to assess the logistics of VMN dissection in the hypothalamus in terms of yielding sufficient material for eventual DPP2 substrate identification. It is highly likely that this substrate is a secreted neuropeptide. Thus, for our initial approach, we propose to use shotgun proteomics to analyze the proteins secreted by the dissected VMN after short culture at 37 C. This work will be performed in collaboration with John R. Yates, 3rd, at Scripps Research Institute in La Jolla, one of the top experts in the field of identifying neuropeptides by this approach. Since the N-terminus controls protein turnover, we expect to see a quantitative difference in the level of a DPP2-subsrate protein in the two strains. In addition, we will look for N-terminal peptides that are modified by DPP2 in wild type, but not mutant mice. Candidate substrates will then be analyzed by in situ hybridization of sections of the VMN. The special strength of this application is the unique expertise of the two PIs, which is highly complementary.